Use of lysophosphatidylethanolamine (18.1) and lysophosphatidylinositol to retard senescence and to enhance fruit ripening

ABSTRACT

The present invention relates to a method of enhancing fruit ripening and stability and of delaying senescence in fruit and other plant tissues. This method consists of applying an effective amount of a lysophospholipid, such as lysophosphatidylethanolamine (18:1) (hereinafter referred to as “LPE (18:1)”) or lysophosphatidylinositol (hereinafter referred to as “LPI”) to the fruit and other plant tissues. Lysophospholipids such as LPE (18:1) and LPI were found to be superior to other lysophospholipids in delaying senescence and in inhibiting phospholipase D, a key enzyme in mediating membrane deterioration during of plant senescence. LPE (18:1) and LPI are naturally occurring and environmentally safe. Their use could replace many environmentally toxic compounds that are currently being used to retard senescence of flowers, fruits and leaves and to enhance fruit ripening.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 application of PCT/US98/23714, Nov. 9, 1998,which claims priority from U.S. Ser. No. 60/064,784 filed on Nov. 10,1997.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded bythe following agencies: USDA AGRICCREE Grant No: 93-37100-8924 and USDAGrant No: 94-34190-1204. The United States has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Various chemical and biological agents are currently being used oncommercially grown fruit to control the timing of fruit ripening. Suchagents can be used for a variety of purposes. One purpose is tosynchronize the ripening of fruit to assist in efficient harvesting offruit from the field. Another purpose is to prevent drop off of fruit sothat fruit remain on the plant until the appropriate ripening timeperiod. Another purpose of fruit ripening agents is to enhance colordevelopment in the fruit so the fruit has a better and more uniformcolor as expected by retail consumers of the fruit. In the UnitedStates, it is current practice for many types of fruit to be treatedwith one or more such agents during the cultivation processes.

Some agents previously used for control of fruit ripening are purelysynthetic agents found to have desired effects on the fruit in question.Unfortunately, due to issues of both potential toxicity andoncogenicity, several such synthetic chemical fruit ripening agents haveeither been banned or had their use sharply curtailed due to commercialor consumer resistance to the products. The most popular agent currentlybeing used to enhance fruit ripening is ethephon, a synthetic compound,which is sold under the name of Ethrel, a trademark of Rhone-Poulenc Ag.Co. (Research Triangle Park, N.C.). Although this agent stimulatesripening, it also causes the fruit to soften. Thus, fruit treated withethephon has a very poor shelf life. There is a critical need for aripening agent which is environmentally safe and which does not causefruits to soften. In addition, consumers are willing to pay a premiumprice for vine ripened fruits. However, vine ripened fruits cannot betransported long distances because these fruits soften and have poorshelf life. Therefore, it would be beneficial to improve the shelf lifeof vine ripened fruits.

There is also a tremendous interest in the plant industry (especially inthe fresh vegetables and cut flower industries) to find anenvironmentally safe product to retard senescence and promote shelf orvase life. Presently, environmentally toxic compounds such as silverthiosulfate are being used to increase the vase life of cut flowers.However, the use of silver thiosulfate is being curtailed because ofenvironmental concerns. Therefore, it is desired to develop alternativesto silver thiosulfate, which are much more likely to be readily acceptedby commercial interests and consuming public.

Lysophosphatidylethanolamines (hereinafter referred to as “LPE”)comprise a group of compounds that have shown promise in controllingfruit ripening, enhancing fruit stability during storage, and increasingthe shelf life of stored fruit. Methods for using LPE purified from egg(hereinafter referred to as “LPEegg”) to enhance fruit ripening andstability are disclosed in U.S. Pat. Nos. 5,126,155 and 5,100,341, whichare incorporated by reference herein. LPE is derived fromphosphatidylethanolamine, a lipid normally found in cell membranes.Phosphatidylethanolamine is a phospholipid with two fatty acid moietieswhich is abundant in egg yolk. The removal of one fatty acid fromphosphatidylethanolamine by phospholipase A₂ yields LPE.

LPE is also naturally present in plant and animal tissue, especiallyrich in egg yolk and brain tissue. It is available commercially fromAvanti Polar Lipids, Inc. (Alabaster, Ala.). There are numerousdifferent fatty acids that can be found in LPE purified from naturalsources. The fatty acids can vary in the length of a chain as well asthe degree of unsaturation. However, the relative efficacy of variousspecies of LPE and also of different kinds of lysophospholipids otherthan LPE in the control of fruit ripening and enhancing fruit stabilityhas not been examined.

SUMMARY OF THE INVENTION

The present invention relates to a method of delaying senescence infruit or plant tissues. The method involves applying to the fruit andother plant tissues, either prior to or after harvest, a compositioncontaining a lysophospholipid and an activating agent. The compositioncontains an amount of a lysophospholipid that is effective in delayingsenescence in fruit and other plant tissues. The preferredlysophospholipid contained in the composition islysophosphatidylinositol and/or lysophosphatidylethanolamine (18:1). Inaddition to containing the lysophospholipid, the composition may alsocontain an activating agent, such as ethanol, tergitol or sylgard 309.

Moreover, the present invention also relates to a method of enhancingthe ripening and stability of fruit. The method involves applying towhole plants before harvest, a composition containing a lysophospholipidand an activating agent. The composition contains an amount oflysophospholipid that is effective in enhancing fruit ripening andstability. The preferred lysophospholipid contained in the compositionis lysophosphatidylinositol and/or lysophosphatidylethanolamine (18:1).In addition to containing the lysophospholipid, the composition may alsocontain an activating agent, such as ethanol, tergitol or sylgard 309.

BRIEF DESCRIPTION THE DRAWINGS

FIG. 1. is a graph showing inhibition of partially purified cabbage PLDactivity by various concentrations of LPE with different acyl chains.

FIG. 2. is graph showing the structural specificity of LPE (18:1) forits inhibition of partially purified cabbage PLD activity.

FIG. 3. is a plot showing inhibition of partially purified cabbage PLDactivity as a function of LPE concentration.

FIG. 4. is a plot showing the effect of substrate concentration on theinhibition of partially purified cabbage PLD by LPE (18:1).

FIG. 5. is a graph showing the effect of different lysophospholipids onpartially purified cabbage PLD activity.

FIG. 6. is a graph showing the relative chlorophyll content of leavestreated with LPA, LPC, LPEegg, or LPI.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of enhancing fruit ripeningand stability and delaying senesence in fruit and other plant tissuesusing lysophospholipids, including, but not limited to, LPE (18:1)and/or lysophosphatidylinositol (hereinafter referred to as “LPI”). Asused herein, the term “lysophospholipids” refers to derivatives ofphospholipids having a single fatty acid removed by phospholipase A₂. Asused herein, the term, “plant tissues” refers to any part or organ froma live plant. Examples include fruit, flowers, roots, stems, hypocotyls,leaves, petioles, petals, etc.

The method of the present invention involves treating fruit and otherplant tissues prior to or after harvest with a composition containing alysophospholipid having the formula:

where R₁ is selected from the group consisting of C₅-C₂₂ acyloxy andC₅-C₂₂ alkoxy group; R₂ is selected from the group consisting ofhydrogen, hydroxyl, C₁-C₅ acyloxy and C₁-C₅ alkoxy group; and R₃ isselected from the group consisting of hydrogen, choline, ethanolamine,glycerol, inositol and serine, wherein R₁ and R₂ are interchangeablewith each other. Preferred compounds having the above-identified formula(1) are LPE (18:1) and LPI.

Preferably, the composition contains an acceptable carrier for thelysophospholipid, such as water. However, other carriers, such asorganic solvents, can be used as well. The composition contains anamount of lysophospholipid that is effective in enhancing fruit ripeningand stability and in delaying senescence in fruit and other planttissues. More specifically, the amount of lysophospholipid in thecomposition can be from about 0.5 to about 1000 mg per 1 liter of thecomposition, preferably from about 1 to about 500 mg per 1 liter of thecomposition, more preferably from about 5 to about 250 mg per 1 liter ofthe composition and even more preferably from about 5 to about 100 mgper 1 liter of the composition. The composition can be applied to thefruit or plant tissues as a spray or simply in liquid form.

In addition to containing the lysophospholipids, the composition canalso contain one or more activating compounds. As used herein, the term“activating compounds” refers to agents that enhance wettability, uptakeand effectiveness of an active ingredient, which is thelysophospholipid. Examples of activating compounds that can be used inthe method of the present invention include ethanol, TERGITOL®(TERGITOL® is a nonylpthenol polyoxyethylene ether. TERGITOL® is aregistered Trademark of Union Carbide Chemicals and Plastics Company,Inc., available from Sigma Chemical Company, St. Louis, Mo.) andSYLGARD® 309 (SYLGARD® 309 is 76% siloxylated polyether and 24% of asurfactant mixture. SYLGARD® is a registered trademark and is availablefrom Dow Corning Co., Midland, Mich.). The activating compounds can bepresent in the composition in the amount of from about 0.05% to about 2%(v/v) of the composition.

The preferred lysophospholipid, LPE (18:1), is a species of LPE havingan 18 carbon fatty acid containing a single double bond. LPE (18:1) hasbeen found to be particularly superior to other species of LPE inpromoting fruit ripening and delaying senescence of fruit and planttissues. LPI has been found to be comparable to LPE (18:1) and superiorto LPEs other than LPE (18:1) in enhancing fruit ripening and indelaying senescence of fruit and plant tissues.

As disclosed in U.S. Pat. Nos. 5,126,155 and 5,110,341, LPE is effectivein enhancing fruit ripening and stability. The exact mechanism by whichthese effects are achieved is only partially understood. It wasdisclosed in U.S. Pat. Nos. 5,126,155 and 5,110,341 that LPE wasobserved to stimulate ethylene production and suppress respiration infruit. It was speculated that these effects might account for theenhanced ripening and stability of LPE-treated fruit. Delayed senescenceof LPE-treated fruit and plant tissues was found to be correlated withreduced leakage of electrolytes through membranes (5). Thus, theinventors suspect that LPE may regulate a key process of membranedeterioration in plant senescence and aging.

Increased leakage of electrolytes during plant senescence has beenascribed to the breakdown of membrane phospholipids (1,2). Reducedleakage of electrolytes in LPE-treated leaves, flowers and postharvestfruits suggests that LPE may protect membrane integrity by inhibitingmembrane lipid degradation (3). Based on the kinetics of release ofvarious lipolytic products in vivo and in vitro, phospholipase D(thereinafter referred to as “PLD”) has been proposed to mediate theselective degradation of membrane phospholipids, which is a rapid andearly event occurring in senescing tissues (4-9). An increase in PLDexpression was observed in senescing leaf tissues and the expression ofPLD was characterized by complex modes including an increase inmembrane-associated PLD, differential expression of PLD variants, andchanges in amounts of PLD protein and mRNA (10).

As described herein, the inventors demonstrate that the lysophospholipidLPE can inhibit the activity of partially purified PLD in a highlyspecific manner in plants. As the following examples below demonstrate,the lysophospholipids LPE (18:1) and LPI are particularly stronginhibitors of PLD. In addition, treatment of plants with LPE (18:1) orLPI is associated with reduced ethylene production. LPI has been foundto be particularly effective in delaying senescence in leaves, asevidenced by the high chlorophyll content of LPI-treated senescingleaves, relative to a control as well as compared to LPE or LPC-treatedleaves. Consequently, lysophospholipids, such as, but not limited to,LPE (18:1) and LPI are particularly attractive agents for delayingsenescence of fruit and plant tissues. The inventors also demonstratethat LPE (18:1) and LPI are particularly effective in enhancing fruitripening and stability.

By way of example and not of limitation, examples of the presentinvention will now be given.

EXAMPLE 1

Specific Inhibition of PLD by LPE (18:1) and LPI

EXAMPLE 1a

Chemicals and Plant Materials

Natural lysophospholipids purified from egg yolk, bovine liver, andsoybean and synthetic LPE with different acyl chains (14:0, 16:0, 18:0,18:1) were obtained from Avanti Polar Lipids (Alabaster, Ala.). Allother phospholipid chemicals and materials used were obtained from Sigma(St. Louis, Mo.). Phospholipids and fatty acid were dissolved inchloroform:methanol:KOH (1N) (95:5:1, v/v). After water was added,organic solvents were expelled by flowing nitrogen gas. Stock solutionconcentrations were adjusted to 1 mM with water before being added tothe PLD reaction mixture. The LPE solution for treating fruit and planttissues was prepared in bulk by sonicating LPE powder suspended in waterwithout the use of organic solvents.

Partially purified cabbage PLD, which has commonly been used forinvestigating the biochemical and physiological aspects of PLD (11,12),was dissolved in 50 mM Tris (pH 8.0) and added to a reaction mixturewith a final concentration of 0.6 μg/ml in order to examine the effectof LPE on PLD activity.

In addition to the partially purified cabbage PLD, the inventors alsoinvestigated the effect of LPE on the activities of membrane-associatedPLD and soluble PLD which were obtained from two plant sources, i.e.cabbage (Brassica oleracea L. Blue Vintage) and castor bean (Ricinuscommunis L. cv. Hale). Castor bean plants were grown in plastic potscontaining a mixture of vermiculite and parlayed (1:1, v/v), which weresubirrigated at 22° C. with Hoagland nutrient solution under cool-whitefluorescent lights (150 μmol min^(−m)m⁻²) with a 14-h photoperiod (10).Cabbage was obtained from fresh market.

EXAMPLE 1b

Tissue Fractionation

Fully expanded leaves from two-month-old castor bean plants and cabbagewere harvested, quickly frozen in liquid nitrogen, and homogenized witha mortar and pestle chilled on ice (13). An extraction buffer containing50 mM Tris-HCl (pH 8.0) 10 mM KCl, 1 mM EDTA, 0.5 mM PMSF, and 2 mM DTTwas added to the powder samples. After grinding for additional 5 min.,the homogenate was centrifuged at 6,000 g for 10 min. to remove debris.The supernatant was centrifuged at 100,000 g for 30 min. to fractionatethe extract into soluble and membrane-associated PLD. The resultantsupernatant was collected as the soluble fraction and the pellet as themembrane fraction. The membrane fraction was washed once with extractbuffer to remove soluble contaminants. The soluble PLD andmembrane-associated PLD samples were added to the reaction mixtures atfinal concentrations of 100 μg/ml and 10 μg/ml, respectively.

EXAMPLE 1c

PLD Activity Assay

The activity of partially purified cabbage PLD was assayed by measuringthe phosphorus content contained in phosphatidylethanol (hereinafterreferred to as “PEOH”) and phosphatidic acid (hereinafter referred to as“PA”) released from the substrate phosphatidylcholine (hereinafterreferred to as “PC”) (13). For this assay, 20 μmol of PC from egg inchloroform was dried under a stream of nitrogen gas. The lipid wasemulsified in 1 ml H₂O by sonication at room temperature. A standardenzyme assay mixture contained 100 mM Mes/NaOH (pH 6.5), 50 mM CaCl₂,0.5 mM SDS, 20 μl substrate (0.4 μmol PC), 1% ethanol and 20 μl PLD in atotal volume of 200 μl (14). The assay mixture was then incubated at 30°C. for 30 min. in the water bath. The reaction was stopped by adding 750μl chloroform:methanol (1:2). Chloroform (200 μl) was added to themixture followed by 200 μl of KCl (2M). After vortexing, the chloroformand aqueous phases were separated by centrifugation at 12,000 g for 5min. The chloroform phase was collected and dried. The dried sampleswere dissolved in 50 μl of chloroform before they were spotted onto aTLC plate (silica gel G). The plate was developed with solvent ofchloroform:methanol:NH₄OH (65:35:5). Lipids on plates were visualize byexposure to iodine vapor. Spots corresponding to the lipid standardsPEOH, PA and PC were scraped into vials and the amounts were quantitatedby measuring phosphorus content as described in Rouser et al. (15).PEOH, the product of transphosphatidylation reaction, was used as theindicator of PLD activity rather than PA, the product of hydrolyticreaction, since the former is not readily metabolized.

The PLD activity associated with the membrane and soluble fractionsobtained from cabbage and castor bean tissues were measured byquantifying the release of radio-labeled PEOH and PA from the substratePC (10). For this purpose, 0.4 μCi of L-3-phosphatidylcholine,1,2-di[1-C¹⁴] palmitoyl (Amersham (Arlington Heights, Ill.)) was mixedwith 20 μmol PC from egg in chloroform. The assay condition and reactionproduct separation were the same as described above. Radioactivity inPEOH, PA and PC scraped from the TLC was quantitated by scintillationspectroscopy.

EXAMPLE 1d

LPE Treatment and Fruit Ethylene Production

Postharvest treatment of fruit tissues with LPEegg (which is purifiedfrom egg and consists mostly of LPE 16:0 and LPE 18:0) has previouslybeen found to retard senescence and enhance shelf life of fruits (3,16).However, the impact of different acyl chains of LPE on fruit senescencehas not been investigated. In the present study, complementary to theeffect of different acyl chains of LPE on PLD activity, the inventorsinvestigated the effect of different acyl chains of LPE on ethyleneproduction of cranberry fruits. Fully ripened cranberry fruits(Vaccinium macrocarpon Ait. ‘Stevens’) were harvested during the fallseason and kept in a cold room. Randomly selected postharvest cranberryfruits (15 berries per sample) were dipped into LPE solutions withdifferent acyl chains (100 μM) for 30 minutes, then air-dried and leftat room temperature (26±2° C.). After two days, berries were incubatedin a sealed glass jar for 24 hours in order to measure ethyleneproduction. Ethylene was quantified with a gas chromatograph equippedwith a flame ionization detector (Shinadzu 9AM, Shimadzu Corporation,Kyoto, Japan) (3).

EXAMPLE 1e

Effect of Lysophospholipids on Leaf Chlorophyll Content

To evaluate the relative efficacy of various species oflysophospholipids in delaying senescence in leaves, treatment solutionscontaining lysophosphatidic acid (LPA), lysophosphatidylcholine(LPC),LPE, LPI, or water were applied to leaves. The chlorophyll contentof each sample was measured by standard methods (10) after an eight daysenescence. The relative chlorophyll contents were expressed as thepercentage of the control.

EXAMPLE 1f

Results and Discussion

Inhibition of PLD Activity by LPE with Different Acyl Chains

The inventors studied whether LPE, a naturally occurring phospholipid,acts a biologically active lipid mediator by inhibiting PLD activity invitro in a specific manner. The inhibitory effects of LPE on partiallypurified cabbage PLD were assayed using PC as substrate. The PLDactivity was inhibited by LPE with different acyl chains at theconcentrations of 40 and 200 μM (FIG. 1). The extent of inhibitionincreased with the length and the unsaturation of acyl chains. LPE withan acyl chain of 18:1 was the most effective inhibitor among the testedspecies and resultant PLD activity was 16% and 11% of the control at theLPE concentrations of 40 and 200 μM, respectively. On the other hand,LPE 14:0, which is seldom present in plant tissues, had very littleeffect. The effects of LPE with other acyl chains including 18:2 and18:3 would be interesting to test but these forms of LPE are notcommercially available at the present time. A dramatic inhibition of PLDby LPE (18:1), as compared to other LPE molecules tested, suggest that aspecific configuration of LPE is needed for this inhibitory effect.

Structural Specificity of LPE (18:1) for Its Inhibition of PLD

The effect of different components of LPE molecules on PLD activity wastested to determine if any structural specificity was necessary for LPEinhibition. The head group (ethanolamine) and acyl chain (18:1 fattyacid) by themselves had no inhibitory effect on PLD activity (FIG. 2).These results indicate that only the intact LPE molecule is capable ofinhibiting PLD, and a loss of any structural components results incomplete ineffectiveness; thus indicating its structural specificity. Infact, phosphatidylethanolamine (PE) had some stimulatory effect on PLDactivity. In the presence of 200 μM PE, PLD activity was 126% of thecontrol (FIG. 2). Since PE is itself a preferential substrate of PLD(17), the increase in PLD activity might be explained by its directstimulating effect on PLD and/or a preferential hydrolysis of PE by PLD.

Dose-Dependency and Kinetics of PLD Inhibition by LPE (18:1)

Inhibition of PLD by LPE was dose-dependent (FIG. 3). LPE (18:1) showeda dramatic inhibitory effect at the 10 μM concentration resulting in 50%activity of the control and a gradual increase of inhibition withincreased concentration up to 200 μM. LPE concentrations of 10 and 200μM reflect 0.5 and 10 mol percents of total phospholipid in reactionmixtures, respectively.

In order to characterize inhibition of PLD, the effects of substrateconcentration on PLD inhibition were analyzed in the presence andabsence of LPE (FIG. 4). Normal assay conditions utilize the saturatingconcentration of substrate (2 mM PC). The inhibitory effect of LPE(18:1) was maintained even at the 4 mM substrate concentration (FIG. 4).The apparent Km for PLD was 1.7 mM and did not change in the presence ofLPE. However, the presence of LPE (18:1) resulted in a dramatic decreasein Vmax (2.9 μmol min⁻¹ mg⁻¹ protein), compared to the control (Vmax of20.0 μmol min⁻¹ mg⁻¹ protein). These results suggest non-competitiveinhibition of PLD by LPE.

In Situ Inhibition of PLD by LPE (18:1)

Since PLD is present not only in a soluble form in the cytosol but alsoin a membrane-associated form, the inventors determined in situinhibition of LPE on membrane-associated PLD extracted from cabbage andcastor bean leaves. Specific activities of membrane-associated andsoluble cabbage PLD were decreased to 59% and 51% of the control in thepresence of LPE (18:1), respectively (Table 1). Membrane-associated andsoluble castor bean PLD activities also decreased to 31% and 30% of thecontrol, respectively. These results indicate that bothmembrane-associated and soluble PLD activities are inhibited by LPE. Theinhibition of PLD associated with membrane and soluble fractions was,however, less pronounced than the inhibition of partially purifiedcabbage PLD by LPE (FIG. 1 and Table 1). This is perhaps due to thepresence of some interfering factors or to presence of the other formsof PLD which are less sensitive to LPE. Partial purification of PLD,therefore, has been suggested to be critical in characterizing theregulatory mechanisms of PLD (18). For this reason, in the presentstudy, the inventors have used partially purified cabbage PLD, which iscommercially available. However, the observed inhibitory effect of LPEon membrane-associated and soluble PLD extracted from leaf tissuessupports the results obtained with partially purified PLD.

TABLE 1

Inhibition of soluble and membrane-associated PLD activities (nmol min⁻¹mg⁻¹ protein) by LPE (18:1). Data are mean ±SE of two separateextractions (duplicate experiments from each extraction) prepared fromcabbage and castor bean leaves.

Soluble PLD Membrane-associated PLD Cabbage Castor Bean Cabbage CastorBean Control 45.2 ± 3.5 10.2 ± 0.1 368.8 ± 6.5 153.8 ± 8.5 LPE 18:1 23.1± 1.6 3.1 ± 0.1 217.0 ± 13.0 47.0 ± 3.6 (200 μM) Ratio (LPE/Control)0.51 0.30 0.59 0.31

Inhibition of Fruit Ethylene Production by LPE with Different AcylChains

Previously, LPEegg (extracted from egg yolk) had been found to delayfruit senescence as indicated by lowered rates of ethylene productionwhen compared to the control (3). Since the inventors found that theinhibitory effectiveness of LPE on PLD was dependent on the length andunsaturation of acyl chain of LPE (FIG. 1), the effects of LPE withdifferent acyl chains on fruit senescence were tested. Cranberry fruitswere treated with LPE with 14:0, 16:0, 18:0 and 18:1 chain lengths, andethylene production by these fruits was monitored. The inhibition ofethylene production increased with acyl chain length and theunsaturation of LPE (Table 2). LPE (18:1) resulted in the most dramaticdecrease (40%) in ethylene production 2 days after treatment.Interestingly, this pattern of inhibition of ethylene production byvarious types of LPE was similar to the pattern of inhibition of PLD byvarious types of LPE (FIG. 1). These results indicate that inhibition ofPLD activity and ethylene production is consistently dependent on theacyl chain length and the unsaturation of LPE. These results suggestthat LPE (18:1) is superior to other LPE species tested in inhibitingPLD and retarding fruit senescence.

TABLE 2

Inhibition of ethylene production in cranberry fruits by LPE (100 μM)with different acyl chains. Values are mean ±SE of three replications.

Control LPE14:0 LPE16:0 LPE18:0 LPE18:1 Ethylene 1.78 ± 1.78 ± 1.65 ±1.27 ± 1.06 ± (nl hr⁻¹g⁻¹) 0.38 0.11 0.05 0.05 0.13 Relative % 100 100.092.7 71.3 59.6

Structurally Selective Regulation of PLD by Lysopholipids

To address whether the inhibition of PLD could occur by a wide range oflysophospholipids, the inhibitory effect of LPE on PLD was compared toinhibition by related lysophospholipids present in plant cells (FIG. 5).Lysophosphatidylcholine (hereinafter referred to as “LPC”),lysophosphatidylglycerol (hereinafter referred to as “LPG”) andlysophosphatidylserine (hereinafter referred to as “LPS”) did notsignificantly affect on PLD activity. However, LPI showed inhibitoryeffects somewhat similar to those of LPE. Whereas, lysophosphatidic acid(hereinafter referred to as “LPA”) significantly increased PLD activity(FIG. 5). For example, at 200 μM concentration LPI and LPA, the PLDactivity was 31% and 169% of the control, respectively. The onlysynthetic lysophospholipid tested in FIG. 5 was LPA. All otherlysophospholipids were from natural sources containing primarily 16:0 or18:0 fatty acids. In addition to LPA (16:0) (FIG. 5), the inventors alsotested LPA (18:1) and found similar results from the two types of LPA.In the present study, LPE but not LPC had a strong inhibitory effect onPLD (FIG. 5). These results indicate that the regulatory effect ofindividual lysophospholipids on PLD enzyme is very specific andstructurally selective.

In addition to LPE, the results suggest that LPI may also be a lipidmediator for retarding senescence in plants. ps Delayed Leaf Senescenceby LPI

The chlorophyll content of senescing leaves treated with LPI was foundto be much higher than leaves treated with LPA, LPC or LPEegg (FIG. 6).This result indicates that LPI is particularly effective in delayingleaf senescence.

In summary of example 1, to the inventors' knowledge, this is the firststudy showing a specific inhibitory regulation of PLD by LPE (18:1) andLPI, which directly target the activity of PLD enzyme. This is asignificant finding since there are no known specific inhibitors of PLDin plants and animals (19). It has also been shown that treatment offruit plants with LPE (18:1) reduces ethylene production more than LPEspecies with shorter acyl chain lengths or a higher degree ofsaturation. Because both ethylene and PLD are associated with senescencein plants, it is reasonably expected that LPE (18:1) and LPI areparticularly effective in delaying senescence in fruit and plant tissuesthan other species of LPE.

EXAMPLE 2

Retarding Senescence and Enhancing the Shelf-Life of Flowers

Flowering spikes of snapdragon (Antirrhinum majus L. cv. Potomac White)were harvested and delivered from a commercial grower overnight (20).Upon receipt, the stem ends of spikes were recut under distilled waterand allowed to rehydrate for 2 hrs. After rehydration, spikes weretrimmed to a length of 40 cm and the leaves on the lower 18 cm of thespike were removed. This prevented leaves from becoming source ofbacterial and fungal contamination in the vase. All spikes were thenpooled and randomly selected for treatment. LPE(18:1), LPI and LPEeggwere prepared in distilled water. Sonication was used to facilitatedissolution of LPE and other lysophospholipids in water.

For the LPE treatment, the cut end of the spikes were held for 24 hr ina solution of LPE at the different concentrations. Thereafter, they weretransferred to distilled water and kept in that water for 3 weeks.Spikes were observed for opening of floral buds and also for symptoms ofsenescence (wilting and browning). If the flower neck became wilted, theflower was considered to be nonmarketable. A spike was consideredmarketable as long as it remained turgid (not wilted) and when more than50% of the florets remained healthy. At the end of the study, the watercontent of spike leaves was determined as an indicator of turgidity andleaf health by measuring the ratio of fresh versus dry weight.

As shown below in Table 3, LPEegg (LPE purified from egg) treatment wasable to retard senescence as compared to control; in the former 37-52%of spikes had wilted while in the control 76% of them had wilted after 7days of treatment. LPE (18:1) and LPI treatment was particularlyeffective in increasing vase life of snapdragon flowers; only 30-39% and15-22% of spikes had wilted, respectively, in these two treatments. LPE(18:1) and LPI not only increased vase life of flower but also improvedthe sensitivity of the flowers to these lipids. For example, 5 mg/L ofLPI and LPE (18:1) yielded more prolonging of flower vase life than did25 mg/L of LPEegg, indicating that LPI and LPE (18:1) are more activeforms among lysophospholipids for the retardation of senescence. Flowerstreated with LPI and LPE (18:1) remained marketable up to 13 days whilewater-treated flowers and LPEegg-treated flowers remained marketable for4 days and 7 days, respectively. Leaf water content of LPE (18:1)-andLPI-treated spikes was higher than that of LPEegg and water-treatedspikes at 18 days after treatment. This data is consistent with theimproved shelf-life of flowers treated with LPE (18:1) and LPI. Thisdata support that LPE (18:1) and LPI are superior to LPEegg.

TABLE 3 Spikes with Wilted Water Content Flower Florets of Leaf after 7days after 18 days Vase Life ** (% of total) (Fresh wt/Dry wt) of SpikesTreatment Mean ± SE* Mean ± SE* (Days) Control 75.5 ± 10.3 5.79 ± 0.18 4 (water) LPEegg  5 mg/L 52.3 ± 9.5 6.77 ± 0.44 — 10 50.0 ± 15.5 — — 2536.7 ± 5.55 —  7 LPE18:1  5 mg/L 34.5 ± 5.5 7.81 ± 0.22 — 10 30.0 ± 5.5— 12 25 38.7 ± 8.5 — — LPI  5 mg/L 21.6 ± 7.0 7.57 ± 0.52 — 10 15.0 ±11.5 — 13 25 17.8 ± 7.5 — — *Date are mean ± SE of two independentexperiments. Each experiment was done with 12 spikes per treatment. **Vase life: Days when > 50% of spikes remained marketable.

Flowering spikes of carnation (Dianthus caryophyllus L. cv. White Sim)obtained from a commercial grower were treated with various lipids asdescribed above for snapdragons. As with the snapdragons, LPI and LPE(18:1) at 25 mg/L were superior to LPEegg and control in prolonging thevase life of carnations (Table 4). LPEsoy (LPE purified from soybean)also gave better shelf-life than LPEegg (LPE purified from egg). LPEsoyconsists of 64% unsaturated LPE, such as LPE (18:1), LPE (18:2), and LPE(18:3) and 30% saturated LPE such as LPE (16:0) and LPE (18:0), and 2%LPI (available from Avanti Polar Lipids, Inc., Alabaster, Ala.), whileLPEegg contains mostly (>94%) saturated LPE such as LPE (16:0) and LPE(18:0). This result supports the inventors' conclusion that LPE (18:1)and LPI are superior to other LPE species in prolonging the vase life offlowers.

TABLE 4 Marketable Flowers after 6 days of treatment (% of total)Treatment *Mean ± SE Control 27.5 ± 3.3 (water) LPEegg 30.8 ± 3.3LPE18:1 41.7 ± 8.3 LPI 44.2 ± 8.3 LPEsoy 41.7 ± 8.3 *Date are mean ± SEof 36 flowers per treatment. (9 flowers/Replications)

EXAMPLE 3

Retardation of Fruit Senescence

Mature green fruits of tomato (Lycopersicon esculentum cv. H9144) wereharvested from three month old plants. Harvested fruits were dipped inthe lysophospholipid solutions indicated in Table 5 below, at theconcentration of 100 mg/L in 1% ethanol for 20 min. The control tomatoeswere dipped in distilled water containing 1% ethol. After dipping, thefruits were stored at room temperature for 3 weeks. Production ofethylene gas was measured 7 days after treatment. The rate of ethyleneproduction by the fruits gradually increased as the fruits started toripen. While mature green at 0 day had no production of ethylene, theuntreated control fruits produced ethylene at the rate of 1.26 nl/g.hr⁻¹after 7 days of treatment (see Table 5 below). LPEegg-treated fruitsshowed production of ethylene similar to control. Whereas, both LPE(18:1) and LPI-treated fruits showed suppression of ethylene production,and this rate was only about half of the control, in LPEegg-treatedfruits, suppression of ethylene production correlated prolonging shelflife of fruits. Consistent with this expectation, the percentages ofrotten fruits after 3 weeks of incubation also indicated that LPE (18:1)and LPI are particularly more effective than LPEegg and the control inextending the shelf life of tomatoes. LPEsoy was found to be better thanLPEegg in terms of prolonging the shelf-life of fruits (rotten fruits24% in LPEegg and 15% in LPEsoy).

TABLE 5 Ethylene Production after 7 days Rotten fruits (nl/g. hr¹) after3 weeks Treatment* Mean ± SE** (% of total) Control 1.26 ± 0.21 37.2LPEegg 1.22 ± 0.22 24.4 LPE18:1 0.70 ± 0.20 7.7 LPI 0.71 ± 0.40 17.5LPEsoy 0.74 ± 0.10 15.0 *All solution were prepared in 1% (v/v) ethanol**Data are mean ± SE of two independent experiments. Each experiment had9 fruits per treatment.

EXAMPLE 4

Retardation of Ethephon-Induced Leaf Senescence

Ethephon, also known as Ethrel, (Ethrel is a trademark of Rhone-PoulencAg. Co. (Research Triangle Park, N.C.)) is an aqueous formulation thatdecomposes to ethylene and is used widely to maximize the yield of ripetomato fruits in once-over harvesting operations. The present inventiondemonstrates that LPE (18:1) is superior to LPEegg and otherlysophospholipids in protecting leaves from ethephon-induced leafsenescence. Tomato plants cv. H9144 were grown in a greenhouse for twoand a half months to serve as sources of leaf samples. Plant weresprayed to runoff with ethephon at 1000 mg/L or with ethephon pluslysophospholipid mixtures as shown below in Table 6. Lysophospholipidsolutions at 50 mg/L were prepared in 1% (v/v) ethanol and mixed withethephon (1000 mg/L). Control plans were sprayed with ethephon alone in1% (v/v) ethanol. Senescence of treated leaves was quantified 10 to 14days after treatment by measuring chlorophyll and protein content.Ethephon-sprayed leaf tissue showed dramatic loss in chlorophyll andprotein content as shown in Table 6 below. LPEegg significantly retardedethephon-induced senescence. LPE (18:1) showed much better retardationof leaf senescence caused by ethephon. LPI had a little retarding effecton ethephon-induced leaf senescence. These results demonstrate that LPE(18:1) works even better than other forms of LPE for this purpose.

TABLE 6 Chlorophyll Content Protein Content (mg/g dry wt) (mg/g dry wt)Treatment* Mean ± SE** Mean ± SE** Ethephon (E) 3.65 ± 0.25  58.8 ± 14.7E + LPEegg 5.88 ± 2.04  81.9 ± 17.6 E + LPE18:1 8.40 ± 2.70 100.0 ± 20.0E + LPI 4.12 ± 0.55  60.0 ± 14.7 *All solution were prepared in 1% (v/v)ethanol. **Data are mean ± SE of three independent experiments. Datawere collected 10 to 14 days after treatment.

EXAMPLE 5

Enhancement of Fruit Ripening

This experiment was conducted to compare the effects of LPEegg, LPE(18:1) and LPI on fruit ripening. Tomato plants cv. H9478 were grown inpots for two and one-half months under fluorescent lights. Whole plantshaving about 10% of their fruits in the ripening stage were sprayed witha solution containing 100 mg/L of different lysophospholipids such asLPEegg, LPE (18:1) or LPI. All solutions contained 1% ethol and 0.05%sylgard 309 (Dow Corning Co., Midland, Mich.) as activating agents.Control plants received distilled water containing 1% ethanol and 0.05%sylgard 309. Fruits were harvested 10 days after treatment and gradedinto green, partial red, and red (indicating full ripening). LPEeggenhanced fruit ripening significantly compared to the control aspreviously disclosed in U.S. Pat. Nos. 5,126,155 and 5,110,341 (seeTable 7). However, LPI and LPE (18:1) were found to be more effectivethan LPEegg. LPI and LPE (18:1) also enhanced fruit stability byprolonging shelf life of post harvest fruits compared to control andLPEegg (see Table 7).

TABLE 7 3 Weeks After Harvest At Harvest Soft Fruits (weight % of total)(non marketable) Treatment* Green Partial Red Red (weight % of total)Control 33.2 13.2 53.6 47.8 LPEegg 22.8 15.9 61.3 36.6 LPE18:1 22.9 11.765.4 33.0 LPI 18.3 11.7 70.1 25.6 *All solution were prepared in 1%(v/v) ethanol and 0.05 and sylgard 309. Spray applications were made 10days before harvest. **Data are average representing three independentexperiments.

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What is claimed is:
 1. A method of delaying senescence in fruit andother plant tissues, the method comprising the step of applying to fruitor other plant tissues a composition comprisinglysophosphatidylinositol, lysophosphatidylethanolamine (18:1) orcombinations thereof.
 2. The method of claim 1 wherein the compositionfurther comprises an activating agent.
 3. The method of claim 1 whereinthe composition is an aqueous solution.
 4. The method of claim 1 whereinthe composition is applied before or after harvest.
 5. The method ofclaim 1 wherein the composition contains an effective amount oflysophosphatidylinositol, lysophosphatidylethanolamine (18:1) orcombinations thereof to delay senescence in fruit and other planttissues.
 6. The method of claim 1 wherein the composition contains fromabout 0.5 to about 1000 mg per liter of lysophosphatidylinositol,lysophosphatidylethanolamine (18:1) or combinations thereof.
 7. Themethod of claim 2 wherein the activating agent is ethanol, a nonylphenolpolyoxyethylene ether or a siloxylated polyether.
 8. A method ofenhancing the ripening and stability of fruits the method comprising thestep of applying preharvest to whole plant tissues a compositioncomprising lysophosphatidylinositol, lysophosphatidylethanolamine (18:1)or combinations thereof.
 9. The method of claim 8 wherein thecomposition further comprises an activating agent.
 10. The method ofclaim 8 wherein the composition is an aqueous solution.
 11. The methodof claim 8 wherein the composition contains an effective amount oflysophosphatidylinositol, lysophosphatidylethanolamine (18:1) orcombinations thereof to enhance ripening and stability of fruit.
 12. Themethod of claim 8 wherein the composition contains from about 0.5 toabout 1000 mg per liter of lysophosphatidylinositol,lysophosphatidylethanolamine (18:1) or combinations thereof. 13.Themethod of claim 9 wherein the activating agent is ethanol, a nonylphenolpolyoxyethylene ether or a siloxylated polyether.